1. Field of the Invention
This invention relates to melanocortin receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of a complementary DNA copy of the messenger RNA (mRNA) of a novel rat melanocortin receptor gene responsive to .gamma.-melanocyte stimulating hormone, .alpha.-melanocyte stimulating hormone and adrenocorticotropic hormone. This receptor has been termed the melanocortin-3 receptor (MC3-R). The invention relates to the construction of eukaryotic recombinant expression constructs capable of expressing MC3-R in cultures of transformed eukaryotic cells, and the production of MC3-R in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells to produce homogeneous compositions of MC3-R protein. The invention also provides cultures of such cells producing MC3-R for the characterization of novel and useful drugs. Antibodies against and epitopes of MC3-R protein are also provided by the invention.
2. Background of the Invention
The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, .alpha.-melanocyte stimulating hormone (.alpha.MSH), and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones, however, are found in a variety of forms with unknown functions, one of which is .gamma.-melanocyte stimulating hormone (.gamma.MSH). Unlike .alpha.MSH or .beta.MSH, .gamma.MSH has little or no ability to stimulate pigmentation (Ling et al., 1979, Life Sci. 25: 1773-1780; Slominski et al., 1992, Life Sci. 50: 1103-1108). However, .gamma.MSH has been shown to have potent pressor, cardioaccelerator and natriuretic actions and acts both in the central nervous system and at the renal afferent nerve (Gruber & Callahan, 1989, Am. Physiol. Soc. 257: R681-R694; Klein et al., 1985, Life Sci. 36: 769-775; Lin et al., 1987, Hypertension 10: 619-627).
The melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain and immune system, and bind to specific receptors in these tissues with a distinct pharmacology [see, Hanneman et al., in Peptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied & Jolles, 1982, Physiol. Rev. 62: 976-1059 for reviews].
A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.
Shimuze, 1985, Yale J. Biol. Med. 58: 561-570 discusses the physiology of melanocyte stimulating hormone.
Tatro & Reichlin, 1987, Endocrinology 121: 1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.
Solca et al., 1989, J. Biol. Chem. 264: 14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactively and photoaffinity labeled MSH analogues.
Siegrist et al., 1991, J. Receptor Res. 11: 323-331 disclose the quantification of receptors on mouse melanoma tissue by receptor autoradiography.
Cone & Mountjoy, U.S. patent application Ser. No. 07/866,979, filed Apr. 10, 1992, disclose the isolation of human and mouse .alpha.-MSH receptor genes and uses thereof (incorporated herein by reference).
Cone & Mountjoy, U.S. patent application Ser. No. 07/866,560, filed Apr. 10, 1992, disclose the isolation of human and bovine ACTH receptor genes and uses thereof (incorporated herein by reference).
Mountjoy et al., 1992, Science 257: 1248-1251 disclose the isolation of cDNAs encoding mammalian ACTH and MSH receptor proteins.
The present invention comprises a nucleic acid comprising a complementary DNA (cDNA) copy of the mRNA of a rat MC3-R gene responsive to .gamma.-melanocyte stimulating hormone. The invention also encompasses the nucleotide sequence of this gene and the deduced amino acid sequence of its cognate protein, a homogeneous composition of the melanocortin-3 receptor (MC3-R), nucleic acid hybridization probes and a method for determining the tissue distribution of expression of the gene, a recombinant expression construct capable of expressing the gene in cultures of transformed eukaryotic cells, and such cultures of transformed eukaryotic cells useful in the characterization of novel and useful drugs.